For the detection of mycolactones in the bacterial culture supernatants, 500 l of well-grownM.ulceranscultures were centrifuged at 13,300 xgto pellet the bacteria, and the supernatant was filtered using sterile 0.22 m syringe filters. systematic selection of a capturing antibody and a reporter molecule, and the optimization of assay conditions, we developed an ELISA that detects common natural variants of mycolactone with a limit of detection in the low nanomolar range. The mycolactone-specific ELISA explained here will be a very useful tool for research around the biology of this macrolide toxin. After conversion into a simple point-of-care test format, the competition assay may have great potential as laboratory assay for both the diagnosis of Buruli ulcer and for the monitoring of treatment efficacy. == Author summary == The macrolide toxin mycolactone is the important virulence factor ofMycobacterium ulcerans, the causative agent of the chronic necrotizing skin disease Buruli ulcer. Mycolactone has cytotoxic activity and is secreted by the mycobacteria, causing tissue necrosis and immunosuppression. For research on Buruli ulcer, there is urgent need for a simple tool to quantify mycolactone. Using the first mycolactone-specific monoclonal antibodies that we have explained previously, we have optimized here an antigen competition ELISA that detects common natural variants of mycolactone at a low nanomolar scale. Sensitivity of the assay is sufficient to detect the toxin inM.ulceransculture supernatants and in the tissue of experimentally infected animals. Converted into a simple point-of-care test format, the competition assay may in future also be suitable as a diagnostic laboratory test for Buruli ulcer. == Introduction == Mycobacterium ulceransis the etiological agent of the chronic necrotizing skin disease Buruli ulcer (BU) that primarily affects children in West and Central Africa [1]. Genomic analyses have shown thatM.ulceranshas emerged from a common ancestor with the fish pathogenMycobacterium marinum[2,3] by acquisition of a virulence plasmid carrying genes that encode polyketide-modifying enzymes and the giant polyketide synthases responsible for the synthesis of the lipid toxin mycolactone [4]. WhileM.marinumoccasionally causes limited granulomatous skin lesions in humans [5], chronicM.ulceransinfections are associated with a much more severe pathology. Mycolactone plays a key role in the chronic necrotizing pathogenesis of BU and, in addition, analgesic and immunosuppressive effects are attributed to the toxin [6]. There is evidence of multiple modes of action of mycolactone, including Fucoxanthin inhibition of Sec61-mediated protein translocation, uncontrolled assembly of actin by binding to the Wiskott-Aldrich syndrome protein (WASP) family, and induction of apoptosis through increased expression of the pro-apoptotic regulator Bim [6,7]. Mycolactone is an amphiphilic molecule, prone to forming aggregates in aqueous solutions [8,9], to binding to soluble proteins [10], and to inserting into lipid bilayers [8,11]. At an air flow/buffer interface, mycolactone has been shown to have surfactant properties with an apparent surface saturation concentration of 1 1 M [8]. Early case detection and quick initiation of antibiotic treatment are currently the important elements of BU control. The disease Fucoxanthin presents in a variety of clinical manifestations, complicating the clinical diagnosis [12]. Laboratory assessments routinely utilized for confirmation of clinical diagnosis include primarily the microscopic detection Rabbit Polyclonal to C/EBP-alpha (phospho-Ser21) of acid-fast bacilli (AFB) andM.ulcerans-specific PCR tests. While microscopythe only diagnostic test that can currently be performed routinely at hospital levelhas limited sensitivity, PCR detecting the insertion sequence Is usually2404is highly sensitive and specific [13]. However, PCR requires sophisticated laboratory infrastructure and well-trained staff and is not reliable without rigid quality control [14]. In resource-poor BU endemic countries, the test is only available at a few research centres, which poses major logistical problems. Therefore, there is urgent need for a simple and Fucoxanthin quick diagnostic test for BU that can be performed at local hospital level or in the field [13]. Mycolactone represents an ideal target for such an assay, since it seems to be unique toM.ulcerans. A mycolactone-specific assay may also be highly suitable for monitoring treatment efficacy and to diagnose relapses, since mycolactone levels in the affected tissue decline during successful specific therapy [15,16]. Mycolactones consist of a core structure, a short C-linked upper side chain, and a longer C5-O-linked lower acyl side chain. Geographical lineages ofM.ulceransproduce different pools of molecular variants of mycolactone, which.